Existence of a low-affinity ATP-binding site in the unphosphorylated calcium-ATPase of sarcoplasmic reticulum vesicles: evidence from binding of 2', 3'-0-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP and -[3H]ATP

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Abstract
ATP-binding sites in the unphosphorylated Ca2+-ATPase of sarcoplasmic reticulum vesicles were titrated with 2'',3''-O-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP (TNP-AMP) or -[3H]ATP (TNP-ATP) in the absence of Ca2+ at pH 7.0 and 0.degree.C by using a centrifugation procedure. In some measurements, the bound TNP-nucleotides were chased with ATP. The data were analyzed by best-fit computer programs as well as by Scatchard plots. The results showed the existence of 1 mol of TNP-AMP binding sites with high affinity (Kd = 7.62 nM) per mole of phosphorylatable sites. The affinity of these sites for ATP (Kd = 10.1 .mu.M) agreed with that of catalytic sites for ATP in the absence of Ca2+. The results further showed the existence of 2 mol of TNP-ATP binding sites with uniform affinity (Kd = 156 nM) per mole of phosphorylatable sites. Half of the bound TNP-ATP was fully chased by low concentrations of ATP. The affinity of this class of the sites for ATP (Kd = 8.9 .mu.M) again agreed with that of catalytic sites for ATP. The other half of the bound TNP-ATP was fully chased only by much higher concentrations of ATP. Thus, the affinity of this class of the sites for ATP (Kd = 791 .mu.M) was much lower than that of catalytic sites for ATP. Similar measurements were performed with sarcoplasmic reticulum vesicles pretreated by N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylenediamine. Although the affinities for TNP-ATP and for ATP were appreciably altered by this pretreatment, the results were essentially the same as those obtained with native vesicles. These results demonstrate that, in the unphosphorylated enzyme, there exists 1 mol of low-affinity ATP-binding sites as well as 1 mol of high-affinity ATP-binding sites (catalytic sites) per mole of phosphorylatable sites.

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